The present invention is directed to the field of inducing resistance in plants to a broad range of pathogens. Specifically, the invention relates to methods of inducing resistance in plants against a variety of viruses, bacteria, and fungal pathogens by expression of a viral polyprotein encoded by members of the potyvirus group of plant viruses or by expression of other suppressors of gene silencing.
The survival of a plant attacked by an invading pathogen depends upon a number of factors. One important factor is how quickly the plant responds and mounts a defense to the pathogen, including the induction of both local and systemic pathways for resistance. Local responses to pathogen attack include callose deposition, physical thickening of cell walls (lignification), synthesis of compounds which exhibit antibiotic activity (e.g., phytoalexins), and the production of protein molecules like cell wall hydrolases (Ward et al., xe2x80x9cCoordinate Gene Activity in Response to Agents that Induce Systemic Acquired Resistance,xe2x80x9d The Plant Cell 3: 1085-1094, 1991; the contents of which is incorporated herein in its entirety).
In addition to mounting a local response, plants may also employ general resistance mechanisms to prevent an infection from spreading or to fight secondary infections from a broad array of pathogens. For example, induction of a hypersensitive response (HR), resulting from the interaction of a specific resistance gene from the plant with the corresponding avirulence gene from the pathogen, results in localized cell death and permits a plant to mount a rapid defense against a pathogenic organism (Dong, Xinnian, xe2x80x9cSA, JA, Ethylene, and Disease Resistance in Plants,xe2x80x9d Current Opinion in Plant Biology 1: 316-323, 1998; the contents of which is incorporated herein in its entirety).
Another general mechanism of resistance, which may be induced either after an HR response or during an active infection, is known as systemic acquired resistance (SAR). SAR refers to a distinct signal transduction pathway which results in the development of a broad-spectrum resistance (for a general review of the SAR pathway, see Chen et al., xe2x80x9cInduction, Modification, and Transduction of the Salicyclic Acid Signal in Plant Defense Responses,xe2x80x9d Proc. Natl. Acad. Sci. USA 92: 4134-4137, 1995; Hunt and Ryals, xe2x80x9cSystemic Acquired Resistance Signal Transduction,xe2x80x9d Crit. Rev. Plant Sci. 15: 583-606, 1996; Neuenschwander et al., xe2x80x9cSystemic Acquired Resistance,xe2x80x9d pgs. 81-106 in Plant-Microbe Interactions, Vol. 1, Chapman and Hall, New York, 1996; and Shirasu et al., xe2x80x9cSignal Transduction in Plant Immunity,xe2x80x9d Curr. Opin. Immunol. 8: in press, 1996; the contents of which are incorporated herein in their entirety).
The accumulation of salicylic acid (SA) in plants is believed to serve as one signal for the onset of SAR, since the removal of SA in transgenic plants expressing salicylate hydroxylase prevents SAR from being established (Dong, 1998). In some plants, studies have demonstrated that SA alone is sufficient for SAR induction. For example, treatment of plants such as tobacco, cucumber, and Arabidopsis with SA, or its functional analogues, induces SAR (Metraux el al., xe2x80x9cInduced Resistance in Cucumber in Response to 2,6-Dichloroisonicotinic Acid and Pathogens,xe2x80x9d pgs. 432-439 in Advances in Molecular Genetics of Plant-Microbe Interactions, Kluwer Academic Publishers, The Netherlands, 1991; the contents of which is incorporated herein in its entirety.)
Tobacco remains one of the best characterized models of SAR. In tobacco, SAR has been shown to provide resistance against seven of nine tobacco pathogens, including infection with fungal, bacterial, and viral isolates (Ryals et al., xe2x80x9cSystemic Acquired Resistance,xe2x80x9d The Plant Cell 8:1809-1819, 1996; the contents of which is incorporated herein in its entirety).
SAR is not the only mechanism used by plants to induce a broad-spectrum resistance. Evidence indicates that other signals may play a role in the induction of resistance against microbial pathogens. For example, two alternative signal molecules, jasmonic acid and ethylene, have been shown to induce resistance, as well as mediate the wounding response in plants (Dong, 1998).
In addition to signal molecules which induce resistance pathways, it is known that plants contain many genes which encode for defense-related proteins. In addition to the genes involved in mediating HR, and encoding signal transduction molecules like SA, genes encoding pathogenesis-related (PR) proteins, such as phytoalexins, and enzymes involved in providing stress protection and repairing tissue damage are important mediators of protection in plants (P. Reymond and E. E. Farmer, xe2x80x9cJasmonate and Salicylate as Global Signals for Defense Gene Expression,xe2x80x9d Current Opinion in Plant Biology, 1: 404-411, 1998; the contents of which is incorporated herein in its entirety).
Although it was previously known that exposure to certain pathogens could induce both local protective responses as well as elicit a state of general resistance to a broad range of plant pathogens, the present invention represents the first example of inducing such general resistance by ectopic expression of a viral polyprotein encoded by members of the potyvirus group of plant viruses. The nucleotide sequence encoding this polyprotein, and responsible for the induction of this generalized resistance in plants, has been identified as the P1/HC-Pro sequence.
Previously, the present inventors demonstrated that the potyvirus P1/HC-Pro sequence could function to suppress post-transcriptional gene silencing in plants, thus providing a method to enhance the expression of either foreign or endogenous genes introduced into plants transformed with this xe2x80x9cboosterxe2x80x9d sequence (U.S. Pat. No. 5,939,541 to Vance et al., the contents of which is incorporated herein in its entirety).
For purposes of this specification, the term xe2x80x9cbooster sequencexe2x80x9d refers to the P1/HC-Pro sequence of a potyvirus, or at least the part of the P1/HC-Pro coding sequence able to induce a generalized resistance in plants transformed with that sequence.
The term xe2x80x9cgenexe2x80x9d or xe2x80x9cgenesxe2x80x9d is used to mean nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any functional portion of such whole RNA or whole protein sufficient to possess a desired characteristic. Genes that are not part of a particular plant""s genome are referred to as xe2x80x9cforeign genesxe2x80x9d and genes that are a part of a particular plant""s genome are referred to as xe2x80x9cendogenous genes.xe2x80x9d The term xe2x80x9cgene productsxe2x80x9d refers to RNAs or proteins that are encoded by the gene. xe2x80x9cForeign gene productsxe2x80x9d are RNA or proteins encoded by foreign genes and xe2x80x9cendogenous gene productsxe2x80x9d are RNA or proteins encoded by endogenous genes.
The initial step in the present discovery of the viral booster sequence was the finding that PVX/potyviral synergistic disease syndrome, characterized by increases in symptom severity and in accumulation of the PVX pathogen, does not require infection with both viruses. This was reported by Vance, et al. in xe2x80x9c5xe2x80x2 Proximal Potyviral Sequences Mediate Potato Virus X/Potyviral Synergistic Disease in Transgenic Tobacco.xe2x80x9d 206 Virology, 583-590 (1995). The synergistic disease is mimicked in plants expressing only a subset of the potyviral genomic RNA and infected singly with PVX. The potyviral region shown to mediate the synergistic disease comprises the 5xe2x80x2-proximal 2780 nucleotides of the genomic RNA, including the 5xe2x80x2-untranslated region (5xe2x80x2-UTR) and the region encoding the potyviral gene products P1, helper component-proteinase (HC-Pro), and a portion of P3. This described potyviral region is referred to herein as the xe2x80x9cP1/HC-Pro sequence.xe2x80x9d
Thus, Vance et al. (1995), identified a disease determinant carried by the potyvirus genome (the P1/HC-Pro sequence), and this disease determinant was shown to mediate the well-known PVX/potyviral synergistic disease. Although the mechanism by which this potyviral sequence mediated the PVX/potyviral synergistic disease was unknown, it was postulated to involve a specific, direct interaction of the potyviral P1/HC-Pro RNA sequence or the encoded potyviral gene products with the genomic RNA or replication proteins of the interacting PVX pathogen. Although the potyviral P1/HC-Pro sequence was found to boost accumulation of the PVX viral structural gene (coat protein) and the accumulation of the PVX viral particle, this enhanced accumulation was thought to be specific for the native PVX genes expressed from the native PVX genome. Furthermore, the enhanced accumulation of PVX coat protein and PVX virus particles was tightly correlated with the perceived detrimental and undesirable increase in disease symptoms.
Accordingly, it would be beneficial if methods of inducing generalized resistance in plants could be developed, especially if the induction of such resistance did not require initial infection with a pathogen. Additionally, it would be especially beneficial if theinduced resistance functioned to protect the plant from a broad spectrum of pathogens, including infection by viral, bacterial, and fungal organisms. The present invention overcomes some of the deficiencies of prior methods to elicit general resistance in plants by using a particular boosting sequence obtained from a potyvirus or by using other sequences encoding suppressors of gene silencing, particularly suppressors of PTGS (such as, for example, the CMV suppressor of PTGS [Brignetti et al;, EMBO J. 17, 6739-6746, 1998; Beclin et al., Virology 252, 313-317, 1998]). Other suppressors which are useful according to the teachings herein are known to those skilled in the art and can be found, for example, in Voinnet, O., et al., Proc. Natl. Acad. Sci. USA 96, 14147-14152, 1999.
It is an object of the present invention to provide methods for enhancing resistance in plants.
Another object of the present invention is to provide methods of enhancing resistance in plants against a broad spectrum of pathogenic organisms.
A further object of the present invention is to provide processes using a particular booster sequence to induce general resistance against infection in a plant.
Yet a further object of the invention is to provide a method of inducing or enhancing resistance in a plant by providing a virally encoded or other suppressor(s) of gene silencing.
These and other objects are achieved by providing a method for inducing resistance in plants by supplying a virally encoded booster sequence comprising the 5xe2x80x2 proximal region of the potyvirus genome, which may preferably include the coding region for P1, helper component-proteinase (HC-Pro), and a small portion of P3. Alternatively, some portion of the booster sequence sufficient to induce resistance may be expressed either individually or fused to other sequences. Additionally, a modified version of the booster sequence, a related sequence from another virus, or any portion or modified version of that related sequence expressed either individually or fused to another sequence can be employed. It is well known in the art to generate fragments or otherwise modified versions of known sequences by, for example, use of such compounds or methods as Bal 31 exonuclease, restriction enzymes, point mutations, or polynucleotide synthesis. The function of such fragments or modified versions can easily be verified by following the steps taught herein and as specifically exemplified below. Such functional fragments or modifications are included within the scope of the term xe2x80x9cbooster sequence.xe2x80x9d The booster sequence induces resistance against a broad spectrum of pathogenic organisms when suppressed in plants, including viral, bacterial, and fungal organisms.
The process of inducing resistance in plants may be carried out by various methods. For example, the booster sequence may be provided to the plant in a variety of ways. It may be provided by infection with a virus that expresses the booster sequence as a native viral gene product during its natural life cycle. Alternatively, the booster sequence may be introduced through use of a transgenic host plant expressing the booster sequence as an introduced gene. The booster sequence may also be introduced using the same viral expression vector utilized to introduce foreign or endogenous genes of interest. A transient expression system may also be employed to temporarily express the booster sequence.
More specifically, the present invention involves a method of inducing or potentiating resistance in a plant by supplying a virally encoded booster sequence comprising the 5xe2x80x2 proximal region of the potyvirus genome, which preferably includes the coding region for P1, helper component-proteinase (HC-Pro), and a small portion of P3. The booster sequence is introduced into plant material, which includes plant cells, plant protoplasts, or whole plants, so that resistance of the plant to a broad range of pathogens is induced or potentiated, even before the plant containing the booster sequence has come into contact with the pathogen. The 5xe2x80x2 proximal region supplied may comprise the coding region for P1, helper component-proteinase (HC-Pro) and a small portion of P3. The portion of the 5xe2x80x2 proximal region may be expressed independently or fused to other sequences.
Transformation of the plant material with the potyvirus booster sequence, comprising the 5xe2x80x2 proximal region, induces or potentiates general resistance pathways in the plant. The induced or potentiated resistance is to a broad range of pathogens, including viral, bacterial, and fungal isolates. For example, in one embodiment, transgenic plants expressing the booster sequence exhibit enhanced resistance to Tobacco Mosaic Virus (TMV). In a similar fashion, plants supplied with the booster sequence also exhibit increased resistance to fungal infections (i.e., blue mold infection, caused by the pathogen Peronospora tacacina).
The booster sequence may be supplied in a number of ways and by various methods well known to those skilled in the art. For example, the booster sequence may be supplied to a plant (or plant material) by co-infection with a potyvirus that expresses the native booster sequence encoded by that potyvirus; introduction via a viral expression vector with the booster sequence being supplied by co-infection with a potyvirus that expresses a nonnative version of said booster sequence; or introduction via a viral expression vector having the gene fused to the structural gene of said viral expression vector.
Alternatively, the booster sequence may be supplied via expression from one or more DNA copies of the booster sequence stably incorporated into the plant genome prior to, during, or after transformation of the plant material with said introduced gene product according to methods well known to those skilled in the art.
In yet another embodiment according to the present invention, the booster sequence may be operatively linked to an expression control sequence to optimize expression. Recombinant DNA in accordance with the present invention may be in the form of a vector (for example, a plasmid, cosmid, or phage). The vector may be an expression vector, having various regulatory sequences to drive expression, or a cloning vector not having regulatory sequences.
As one of skill in the art of molecular biology will recognize, the booster sequence may be supplied in a number of ways to a host plant. Various teachings may be consulted to determine how to make transgenic plants containing the booster sequence, without undue experimentation. In particular, the following references may be consulted; the contents of which are hereby incorporated by reference in their entirety; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1989; and Gee et al. in Huber and Carr, xe2x80x9cMolecular and Immunologic Approaches,xe2x80x9d Futura Publishing Co., New York, 1994.